Figures

Abstract

After entry into target cells, retroviruses encounter the host restriction
factors such as Fv1 and TRIM5α. While it is clear that these factors target
retrovirus capsid proteins (CA), recognition remains poorly defined in the
absence of structural information. To better understand the binding interaction
between TRIM5α and CA, we selected a panel of novel N-tropic murine
leukaemia virus (N-MLV) escape mutants by a serial passage of replication
competent N-MLV in rhesus macaque TRIM5α (rhTRIM5α)-positive cells using
a small percentage of unrestricted cells to allow multiple rounds of virus
replication. The newly identified mutations, many of which involve changes in
charge, are distributed over the outer ‘top’ surface of N-MLV CA,
including the N-terminal β-hairpin, and map up to 29 Ao apart.
Biological characterisation with a number of restriction factors revealed that
only one of the new mutations affects restriction by human TRIM5α,
indicating significant differences in the binding interaction between N-MLV and
the two TRIM5αs, whereas three of the mutations result in dual sensitivity
to Fv1n and Fv1b. Structural studies of two mutants show
that no major changes in the overall CA conformation are associated with escape
from restriction. We conclude that interactions involving much, if not all, of
the surface of CA are vital for TRIM5α binding.

Author Summary

Host restriction factors such as TRIM5α are important for preventing cross
species transmission of a variety of retroviruses. They act to block viral
replication but their mode of virus recognition is poorly understood. To address
this question we have developed a procedure for isolating viruses that replicate
in the presence of restriction factors. Analysis of these viruses shows that
individual mutations across the entire surface of the viral capsid molecule can
relieve restriction. Escape from TRIM5α of one species does not necessarily
lead to escape from another. It seems likely that restriction factor recognition
involves extensive weak contacts between factor and virus. We suggest that this
represents an important design feature in a system that recognizes multiple
pathogens.

Funding: This work was supported by the UK Medical Research Council; file references
U117512710 (JPS) and U117565647 (IAT). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the
manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Mammalian cells show different susceptibilities to retrovirus infection. Cells from
mice exhibit different susceptibility to murine leukaemia virus (MLV) dependent on
their genetic backgrounds; cell lines from different primates show defined patterns
of lentiviral replication. In many cases, these variations are due to the presence
of cellular proteins referred as restriction factors. The susceptibility to MLV
infection in mouse cells is determined by a restriction factor called Fv1; different
alleles of Fv1 restricting different strains of MLV. Fv1n restricts
B-tropic MLV (B-MLV) but not N-tropic MLV (N-MLV) infections, whereas
Fv1b does the reverse [1], [2], [3]. In primate cells, lentivirus and N-MLV are restricted by
a second restriction factor, TRIM5α [4], [5], [6], [7], [8]. TRIM5 is a member of a large
family of tri-partite motif proteins that contain RING,
B-box and
coiled-coil motifs, sometimes
referred to collectively as an RBCC domain [9], [10]. The α isoform of TRIM5
(TRIM5α) also has an additional large B30.2 or PRYSPRY domain at its C-terminus
[11], [12], important for
recognizing the viral target [13], [14], [15], [16]. Both Fv1 [17], [18] and TRIM5α [5], [19] target the
viral capsid protein (CA), blocking retrovirus replication after viral entry into
the target cell. Restriction by TRIM5α, however, is clearly distinguished from
that by Fv1 in that TRIM5α-mediated restriction normally occurs before or during
reverse transcription [5], [20], whereas Fv1-mediated restriction occurs after the
completion of reverse transcription but prior to viral integration [21], [22].

One of the most surprising features of TRIM5α restriction is the ability of
TRIM5αs from different species to recognize and restrict multiple unrelated
viruses often across different retroviral genera. For example, TRIM5α from the
cotton-top tamarin can recognize at least one member from each of the lentivirus,
gammaretrovirus, betaretrovirus and spumavirus genera and rhesus monkey (rh)
TRIM5α will restrict both HIV-1 and N-MLV [23], [24], [25]. Although it is clear that Fv1 and
TRIM5α target viral CA, in the absence of structural information the recognition
process remains poorly defined. However, it appears likely that single-site binding
is weak and that a productive interaction required for restriction is multivalent
and dependent on the spacing of CA monomers/hexamers in the viral core [26], [27], [28].

One approach to investigating the problem of restriction specificity has been through
genetic characterisation of naturally occurring or experimentally induced variants
in CA that alter virus tropism [29]. In this way amino acid residues involved in the
interaction with restriction factors have been identified. In the case of
Fv1-mediated restriction of MLV, it is known that several surface-exposed amino
acids in the region between α-helices 4 and 6 in CA, which include amino acids
at positions 82, 92–94, 110, 114 and 117, can change the tropism of MLV [30]. Among these
positions, the amino acid at position 110 is the main determinant, since
substitution of amino acid at position 110 (Arg for N-tropic and Glu for B-tropic
MLV) can switch the tropism of MLV [31]. Structural studies show that amino acids in positions
110, 114 and 117 are located in α-helix 6, residue 82 is in the loop between
α-helices 4 and 5, and amino acids 92–94 are located in α-helix 5
[32].
Therefore, the region between α-helices 4 and 6 might be considered a
‘Fv1-binding pocket’ [27]. A number of amino acid substitution experiments have
also suggested that this α4–α6 region may also contribute to
TRIM5α-mediated restriction of N-MLV. These include an R110E mutation that
confers resistance of N-MLV to restriction by human (hu) TRIM5α and an E110R
mutation in B-MLV that results in susceptibility to restriction by huTRIM5α
[19]. Other
substitutions in CA at positions 82, 114 and 117 alter the TRIM5α species range
able to restrict B- and N-MLV [8] and domain-swap experiments suggested that the equivalent
region in HIV and other lentiviruses also contains the host-range determining
segments [33],
[34], [35], [36].

In this study, replication competent N-MLV was passaged in rhTRIM5α-positive
cells to search for escape mutations in sites important for restriction and in the
context of a natural infection. We isolated a number of N-MLV escape mutants,
including substitutions at the previously identified positions 82 and 114. However,
of particular interest is a new class of mutations including G8D, L10W and V116D
that were discovered. They map outside the putative Fv1 binding pocket and display
subtle structural changes. In these instances, escape from restriction by
rhTRIM5α was conferred, but restriction by huTRIM5α was not affected. These
observations have defined new regions of N-MLV CA that control virus growth in the
presence of TRIM5α, revealed that the restriction factor-CA interface is larger
than previously thought and shown that subtle changes in CA structure can have
profound effects on TRIM5α susceptibility.

Results

In an initial attempt to isolate MLV variants resistant to TRIM5α-mediated
restriction, Mus dunni tail fibroblasts (MDTF), MDTF expressing YFP
and huTRIM5α (MDHu) or rhTRIM5α (MDRh) were inoculated with different
dilutions of replication competent N-MLV (Stock A) and the infected cells were
passed repeatedly. On each passage, the culture supernatant was harvested and
reverse transcriptase (RT) activity in the filtered culture supernatant measured
(Figure 1). After only three
days high RT levels were measured in restriction factor-negative cells, even when
the target cells were initially infected with low titre (1∶100 diluted virus)
(Figure 1A). When
restriction factor-negative cells were infected with the high titre undiluted virus,
the RT activity decreased sharply at 6 days post infection (d.p.i.) but later
recovered, due to virus-mediated cytopathic effects. In cells expressing
huTRIM5α N-MLV replication was restricted completely, even when undiluted virus
was inoculated (Figure 1B). By
contrast, cells expressing rhTRIM5α did not prevent replication of high titre
virus, and, although the time of RT release was delayed, maximal levels were similar
to those seen with MDTF (compare Figure
1C and Figure 1A). No
virus was recovered from MDRh infected with 1∶10 or 1∶100 diluted N-MLV
(Figure 1C).

Taken together, these data imply (a) huTRIM5α restriction of N-MLV is more potent
than that of rhTRIM5α and (b) rhTRIM5α restriction can be overcome by high
levels of wild-type N-MLV in a rapid manner, presumably by saturation. However,
under these conditions, an extended period of virus replication against a background
of restriction does not seem to occur leaving little opportunity for escape mutants
to arise. To maximize the chances of generating and recovering escape mutants we
decided to focus on searching for escape mutants from rhTRIM5α using low input
virus to avoid restriction factor saturation. To allow continued virus replication
under selective conditions a mixture of MDTF and MDRh was infected.

Generation and selection of N-MLV escape mutants from rhesus
TRIM5α

Low titres of N-MLV were used to infect a mixture of 90%
TRIM5α-positive and 10% TRIM5α-negative cells. Virus spread was
monitored by fluorescence staining using a monoclonal antibody directed against
the viral p12 protein. As shown in Figure 2A, virus-infected cell populations were clearly
distinguishable from virus-negative cells both in YFP-negative
(TRIM5α-negative) and -positive (TRIM5α-positive) populations. Following
infection of the first mixed culture, a small fraction of the YFP-positive cells
appeared virus-positive as early as 3 d.p.i.; the virus spread into about
30% of the YFP-positive cell population within 13 d.p.i. and 60%
by 18 d.p.i. (Figure 2B
iv-vi). The culture supernatant was then collected, filtered, diluted
tenfold and passed again in mixed cell culture for a further ten days
(2nd culture). To select mutant viruses, the filtered and diluted
culture supernatant from the second mixed cell culture was inoculated onto
100% MDRh cells (the 3rd and 4th cultures). In
contrast to wild type N-MLV, which at 1∶10 dilution had shown no virus
expansion during the first 18 days of MDRh culture (Figure 1C), the virus from the second culture
took 7 days to grow in 100% MDRh cells in the third culture (Figure 2B). This was not a
result of different levels of viral input because virus with RT activity of 20
mU was inoculated in the third culture compared to 514 mU RT activity in the
first inoculum. The virus at the end of the third culture was stored and
referred to as rhesus TRIM5α-escape mutant 1 (REM1).

Escape mutants were derived by growing N-MLV on cell cultures containing
mixed populations of TRIM5α-expressing and non-expressing MDTF
cells. Virus spread was monitored by staining with an antibody specific
to p12; the presence of TRIM5α was indicated by YFP. (A)
Representative FACS profiles. (i) uninfected MDTF cells, (ii) MDTF cells
3 days post infection (d.p.i.) with N-MLV stained with anti-p12 (iii)
– (vi) panels showing 90% TRIM5-positive cells at 0 d.p.i.
(iii), 3 d.p.i. (iv), 13 d.p.i. (v) and 20 d.p.i. (vi) with N-MLV. Note
that YFP-positive cell population shifted up into the dual-positive
fraction. (B) Emergence of escape mutant in the culture. The percentage
of p12 viral antigen positive cells in the culture was plotted against
the time, d.p.i. Cells were passaged every 3 days for three weeks before
cell-free virus passage to establish the next culture. The first and
second cultures were performed in the mixed cell culture of
TRIM5-positive cells with 10% TRIM5-negative cells, followed by
the third and forth cultures with 100% TRIM5-positive cells. In
the mixed cell cultures, p12-positive cell percentages were calculated
for the YFP positive cells. Input RT activities: 1st culture
514 mU; 2nd culture 1151 mU; 3rd culture 20 mU;
4th culture 712 mU.

This procedure was repeated a further three times starting with N-MLV Stock A
yielding REM2-4. In each experiment a similar time course was observed for
isolation of a virus capable of unrestricted growth in the presence of
rhTRIM5α. These results clearly demonstrate the utility of unrestricted
refugia in the isolation of escape mutants from rhTRIM5α by allowing growth
against a background of restriction.

The primary target for Fv1 and TRIM5α restriction is thought to be the viral
CA protein. It was thus expected that escape mutations would map to the capsid
so the nucleotide sequences of the capsid region of the viral
gag gene was determined by direct sequencing of viral cDNA
amplified from viral RNA in the culture supernatant. These data revealed that
REM1 had acquired a single nucleotide substitution from T to G at nucleotide
1309 (AKV numbering) [37], which is predicted to produce a leucine to
tryptophan substitution at amino acid position 10 in CA. The same L10W mutation
in CA was seen in four independent experiments (isolates REM1-4) and no other
mutations were detected in the capsid gene. This suggested that a small
population of viruses carrying the L10W mutant may already exist in the virus
stock prepared from the culture supernatant of chronically infected cells (Stock
A) and the mutant was selected in the course of the virus passage experiments.
As a first test of this hypothesis, the capsid gene of Stock A virus was
amplified by RT-PCR and cloned. No sequence variation was observed in 50
independent clones (all 50 clones yielded sequences identical to N-MLV derived
from pWN41) indicating that any minority species can only be present at very low
levels.

We next derived a second stock of virus by transfection of 293T cells with
self-ligated pWN41 provirus followed by three cell-free transfers to MDTF cells
(Stock B). Two independent experiments with the new N-MLV stock following the
same protocol used to isolate REM 1-4 resulted in the isolation of potential
escape mutants REM5, 6. The only change in the capsid genes in these two viruses
was a single T to A change at nucleotide 1627, giving rise to a V116D mutation.
Four further experiments with freshly prepared Stock Bs gave rise to REM7-10;
three carried single mutations resulting in L10W, H114N and H114R changes
whereas REM10 carried three mutations causing L4S/A95D/S202G alterations.

A third set of experiments used Stock C, which was recovered directly following
transfection of human 293T cells without passage through murine cells and should
therefore have no mutations associated with reverse transcription (pWN41 encodes
an ecotropic virus which will only infect murine cells), led to the isolation of
REM11-15. Sequencing studies showed that these viruses carried mutations
resulting in the following changes: G8D, N82D (twice), E92K and H114D.

To confirm the importance of these mutations in escape from rhTRIM5α they
were introduced by Quikchange PCR into the N-MLV vector, pCIG3N and the
resulting viruses tested for growth with different restriction factors. On the
basis of these analyses (Figure
3 and Supplementary Figure S1), the escape mutants could be
placed into two groups depending on the degree of residual sensitivity to
rhTRIM5α. One group, typified by L10W and comprising L10W, H114D, H114N,
H114R and E92K showed equivalent titration curves in the presence and absence of
rhesus TRIM5α (though E92K grew poorly compared to wild type N-MLV). The
second group, typified by V116D, contained in addition G8D and N82D. This group
showed slightly reduced infectivity in the presence of rhTRIM5α. Two of the
single amino acid changes present in the triple mutant, L4S and A95D, gave rise
to viruses that would fall in the second group but together show complete
resistance. The S202G change appeared to have no effect on restriction, as might
be expected for a residue in the (interior) C terminal domain of CA. Perhaps
surprisingly, only one of the mutations that result in escape from rhTRIM5α,
E92K, affects restriction by huTRIM5α. Furthermore, three of the mutations
that give rise to resistance to rhTRIM5α, G8D, H114D and V116D confer
sensitivity to Fv1n while maintaining Fv1b susceptibility.
These data are summarized in Table 1.

Since mutations at positions 10 and 116 have not previously been reported to
affect interactions with Fv1 or TRIM5α we next set out to explore the
properties of the L10W and V116D mutations. To examine the interaction between
viral cores carrying the L10W and V116D mutations and rhTRIM5α, we tested
whether such cores would resist rhTRIM5α mediated disassembly using the
previously described fate-of-capsid assay that follows the premature capsid
disassembly thought to occur in an infected cell following virus encountering
TRIM5α [38]. As expected, wt N-MLV cores were pelleted in
TRIM5α-negative cell extract (Figure 4, lane 1) and B-MLV capsid cores, but not wt N-MLV, L10W and
V116D mutant capsid cores, were pelleted when huTRIM5α-expressing cells were
infected (Figure 4, lanes
2–5). This result was consistent with the observations that huTRIM5α
restricted wt N-MLV, L10W and V116D mutant viruses, but not B-MLV. In contrast,
in the presence of rhTRIM5α, B-MLV and the L10W and V116D N-MLVs were
pelleted, but wild type N-MLV was not detected in the pellet fraction (Figure 4, lanes 6–9).
Therefore, L10W and V116D mutant viruses escape disassembly of capsid core
caused by rhTRIM5α, an observation consistent with the lack of restriction
of these mutant viruses by rhTRIM5α and emphasizing the species-specific
nature of escape by L10W and V116D mutants from TRIM5α.

Restriction profiles of L10 mutants and V116D by primate TRIM5αs

The selectivity difference of rhesus and human TRIM5αs prompted us to carry
out a more detailed survey of the restriction profiles. Firstly, as only the
L10W mutation was isolated through the in vivo passaging
experiments we wondered what the effect of other residue types at position 10 in
N-MLV CA might have on restriction factor sensitivity. To address this
possibility, random amino acids were introduced at position 10 by PCR
mutagenesis. Using this procedure fourteen of the possible nineteen
substitutions and wt silent codon changes were recovered. The restriction data
for rhTRIM5α are shown in Supplementary Figure S2
and summarized in Table 2.
Out of the entire panel only two additional mutants, L10H and L10K, giving
complete escape were identified. Two other substitutions (L10P and L10V)
resulted in wt restriction phenotype; another gave non-infectious virus (L10R),
whereas the remainder display intermediate phenotypes with restriction by
TRIM5α at low virus titre but with only moderate restriction at higher virus
titre. The variable phenotypes produced in this experiment highlight the
importance of MLV residue 10 suggesting a direct involvement/interaction of this
part of CA with rhTRIM5α in restriction of N-MLV but do not suggest a direct
correlation between restriction activity and the size or charge of the amino
acid residue at position 10.

Next we tested the ability of a previously described panel of primate TRIM5αs
to restrict the L10W, L10K, L10H and V116D mutant viruses [24]. Wild type N-MLV was
restricted by all the ape and old world monkey TRIM5αs tested as well as
some of the new world monkey TRIM5α including from Cotton-top tamarin and
Brown capuchin (Table 3
and Supplementary Figure S3). All the L10 escape mutants were
also restricted by ape TRIM5αs, which was perhaps not unexpected given the
observation that huTRIM5α restricted the L10W mutant virus. However, among
old world monkey TRIM5αs, only African green monkey TRIM5α restricted
the L10 mutants and in each case these mutations also resulted in escape from
restriction by the capuchin factor. On the other hand, the restriction profile
of the V116D mutant virus was rather different. It was not restricted by
orang-utan TRIM5α but was restricted by sooty mangabey and Brown capuchin
TRIM5αs. These observations showed that whereas the viruses with mutations
at position 10 showed a similar phenotype in terms of restriction by TRIM5α,
the V116D mutant exhibited a different restriction profile by primate
TRIM5α. These results imply that capsid recognition by TRIM5αs of
different species may involve interactions with different amino acids on CA.

Involvement of B30.2 V1 and V2 regions in N-MLV CA recognition

These species differences in restriction profiles of the escape mutants allowed
us to use a previously described series of chimeric human/macaque TRIM5αs
[16], to ask
which of the V1, V2 and V3 variable regions of the TRIM5α B30.2 domain are
important for CA binding (Figure
5, Supplementary Figure S4). Each chimera was designed to
contain a different combination of the variable regions of the B30.2 domain
(Figure 5). Both human
and rhesus TRIM5α restrict N-MLV and so not surprisingly MDTF cells stably
expressing each of the eight chimeric TRIM5αs restricted wild type N-MLV.
The position 10 mutants (L10W, L10K and L10H) show identical patterns of
sensitivity to the chimeric constructs, being unrestricted by HR4, RH3 and RH4,
but restricted by the other chimeric TRIM5α, suggesting that when either the
V1 or V2 region is derived from huTRIM5α, the mutants are restricted,
whereas when both V1 and V2 regions were derived from rhTRIM5α, the mutant
N-MLV was not restricted. The V116D escape mutant also displays an identical
profile to that of the position 10 mutants, supporting the idea that both V1 and
V2 regions are involved in TRIM5α-mediated restriction of MLV.

The left half of the figure shows schematic views of previously described
[16]
chimeras between human (white shading) and rhesus macaque (gray shading)
TRIM5α. The right side summarizes restriction data of mutant viruses
by these TRIM5α. For a full panel of titration curves, see
Supplemental Data (Figure S4). Representative results
are shown from one of three independent experiments. MDTF cells were
transduced with chimeric TRIM5α and cultivated for more than 4 weeks
until cells reached the steady expression level of TRIM5α. YFP
expression was examined 4 weeks after transduction and more than
95% of total cells expressed YFP. TRIM5α-expressing cells
were infected with mutant viruses and the ratio of infected to
non-infected cells was calculated in the YFP-positive cell fraction.
+; restricted more than 10-fold compare to wild type, −;
restricted less than two-fold compare to wild type, and ±;
partially restricted.

The effect of β1-β2 mutations on HIV-1 susceptibility to Rhesus
TRIM5α

The N-terminal domain of HIV-1 and MLV CAs share very similar structures [32], including
the β-hairpin region. It therefore seemed possible that this region might
affect sensitivity of HIV-1 to rhesus TRIM5α. To address this question, each
amino acid between position 5 and 12 on HIV-1 capsid, which form the N-terminal
β-hairpin loop, was substituted with a lysine residue. In addition, the
methionine residue at position 10 of HIV-1 capsid was substituted randomly by
mutagenesis PCR. However, although several of these mutations severely impaired
Gag function, none of the viable HIV-1 mutants examined in this study, escaped
rhTRIM5α (Supplementary Figure S5), implying that unlike MLV, the
N-terminal β-hairpin loop of HIV-1 capsid does not seem to affect the
restriction activity of TRIM5α. Based on these results, the ways by which
rhTRIM5α recognizes viral capsid seems to be different between HIV and
MLV.

Structure of N-MLV CA carrying the L10W mutation

On inspection of the structure of the MLV CA-NtD (Mortuza et al., 2004) it was
apparent that although the side chains of both residues 10 and 116 were surface
exposed on an assembled capsid structure, neither lay in the α4–α6
region, previously proposed as the Fv1 binding site (Mortuza et al., 2008).
Residue 116 was located on the second turn of α6 on the face opposite the
α4–α6 region while residue 10 was even more remote, located on the
N-terminal β1–β2 hairpin. To further examine how the L10W mutation
might effect rhTRIM5α restriction of MLV we determined the crystal structure
of the CA-NtD of this variant at 2.0Å resolution. The structure has been
refined to a final Rwork/Rfree of
17.4%/24.4% respectively and has excellent geometry with only 3
residues in the additionally allowed region of the Ramachandran plot (See Table 4 for details of
crystal parameters, phasing and data refinement statistics).

The structure of L10W was typical of a retroviral CA-NtD consisting of an
N-terminal β-hairpin and a core bundle of six α-helices. A comparison
with the native MLV CA-NtD structure (Figure 6A) revealed an essentially identical
backbone conformation. Pairwise comparisons of L10W with wt N-MLV CA-NtD gave an
RMSD of 0.6Å across equivalent Cα atoms. However, minor deviations
were seen at the top of helix-6 and in the loops and side chains on the surface
of the molecule. In particular, the L10W mutation was clearly visible in the
electron density (Supplementary Figure S6) and was located on the inward
facing side of strand β2 adjacent to the α5-α6 loop and the top of
helix α6.

A) Structural superimposition a monomer of the CA-NtD from
N-MLV (grey) and that of N-MLV(L10W) (blue), side (upper) and top
(lower) views are shown. Structures are displayed in cartoon
representation, secondary structure elements are labeled.
B) Groove between β1–2 and α5–α6 in
wt N-MLV. L10 in the base of the groove is shown in red. R112, T107, and
Q9 lining the grove are shown as sticks. A transparent molecular surface
is shown over the molecule same orientation as A.
C) W10 and R112 block the groove in the structure of
N-MLV L10W. The position of V116 on α6 is also shown.

Examination of the wt N-MLV capsid surface in the proximity of L10 (Figure 6B) reveals the
presence of a shallow channel between the β1–β2 hairpin and
residues at the top of α6 and in the α5–α6 loop. The channel
walls are formed by side-chains of M4 and Q9 from the β1–β2
hairpin together with R112 and T107 from the α5–6 loop. At the bottom
of the channel the aliphatic L10 side-chain forms the base. However, in the L10W
structure (Figure 6C) the
much bulkier tryptophan side-chain protrudes into the channel along with R112
guanidinium moiety. Together they adopt a near planar arrangement reminiscent of
a similar interaction seen in the structure of PSIV capsid [39]. Significantly, this
side-chain configuration in L10W reduces the depth of the
(β1–β2)-α6 channel by some 2.3Å, effectively closing the
cavity completely. To test the importance of the W10 – R112 interaction
for restriction, the R112 amino acid was mutated to an alanine in presence of
both L10 and W10. Both viruses were susceptible to restriction by rhTRIM5α
(Supplementary Figure S7). The observed structural change thus provided a simple
potential explanation for the rhTRIM5α escape phenotype suggesting that in
wt N-MLV the (β1–β2)-α6 channel constituted at least part of
the rhTRIM5α binding site and that by blocking this channel in L10W the
interaction with rhTRIM5α was abolished or sufficiently diminished to allow
the virus to evade restriction.

Discussion

To better understand the interaction between TRIM5α and its viral target, we set
out to isolate a panel of novel N-MLV escape mutants. Noting that direct inoculation
of cells expressing a given restriction factor led, depending on the initial virus
titre, either to rapid break-through of the block in replication or the absence of
replication (Figure 1), we
devised a procedure allowing replication against a background of restriction. We
reasoned that the infection of a mixture of restricting and non-restricting cells
would allow both multiple rounds of unrestricted replication as well as selection
for viruses that were unrecognized by restriction factor. Experiments of the kind
shown in Figure 2 illustrate the
utility of such an approach and have resulted in the isolation of a number of MLV
mutants that escape restriction by rhTRIM5α, apparently by escaping premature
capsid disassembly (Figure 4).
The lack of variation in mutants obtained with Stock A compared with that from
Stocks B and C points to the possible accumulation of variant viruses in
multi-passaged virus stocks even in the absence of overt selection or apparent
sequence variation and emphasize the need for using freshly recovered virus in such
studies.

β-hairpin mutants extend the MLV CA-restriction factor interface

The mutations identified by these passaging experiments together with in
vitro mutagenesis studies all lie on the outer “top”
surface of MLV CA (Figure 7)
and can be divided into two groups. One group (Class-I) comprises N82D, E92K,
A95D, and H114D/R/N, mutations that are within the previously defined Fv1
binding pocket in the loop region between helices 4 and 6 of CA [27], [30]. These
mutations result in escape from rhesus TRIM5α mutations at these positions
can affect susceptibility to Fv1. The other escape mutants, L4S, G8D, L10W,
L10K, L10H and V116D are the founder members of a new group (Class-II) located
on the opposing side of the central α6 helix and in the
β1–β2 N-terminal
β-hairpin. Residue 110 (R in N-MLV and E in B-MLV) is located
on the top of α6 and is often referred to as the major specificity
determinant of restriction as it was the first residue shown to determine
susceptibility to Fv1 or TRIM5α, [8], [19], [31]. Our data now add further
weight to the significance of residue 110 revealing that it is located at the
centre of an extended restriction factor binding-site. This site encompasses the
entire CA top surface from N82 across the region containing Class-I mutants to
residues 8 and 10 in the β-hairpin. The G8D and mutations at
L10 are of particular interest as they are located in a region of CA, the
N-terminal β1–β2 hairpin, where
mutations affecting the interaction of N-MLV with TRIM5α or Fv1 have never
before been reported. The structure and properties of the L10W mutant (Figure 6) and the effect of
the R112A change (Supplementary Figure S7) strongly suggest that the
(β1–β2)-α6 channel present on CA
forms part of the rhTRIM5α binding site. The observation that G8D and V116D
cause susceptibility to Fv1n further suggests that the previous
defined Fv1 binding pocket must be extended to include this region.

Determinants of MLV restriction, side (lower) and top (upper) views of
the N-MLV CA-NtD structure are displayed. The protein backbone is shown
in cartoon representation together with the semi-transparent molecular
surface. Residues involved in restriction factor specificity are shown
as sticks. Determinants of N-B tropism are shown in yellow. Leu 10 that
when substituted by Trp, Lys or His confers resistance to rhTRIM5α
is shown in red. V116 that when substituted by Asp confers resistance to
rhTRIM5α and gives rise to Fv1n sensitivity is shown in blue.

It is noteworthy that residues 10 and 82 are 29 Å (Cα-Cα) apart on
either side of the exposed surface of CA monomers making up the core [32]. Moreover
restriction sensitivity is affected by a number of amino acids (e.g. 92, 95,
110, 114, 116, 117) that are distributed between these two positions, covering
much of the viral core that is exposed to cellular factors after entry into the
cytoplasm of newly infected cells (Figure 7). Many of these residues posses poorly ordered side chains
so their contribution to αrestriction factor binding seems most likely
through side chain charge-charge or hydrophobic interactions at the molecular
interface. Indeed many of the escape mutations we have isolated involve
introduction or loss of charged amino-acids. Further, in cases of mutations
affecting tropism for which we have detailed structural information, R110E [27] and L10W
(this paper) there is no evidence for major change in the overall conformation
of CA, indicating that mutations in the N terminal β-hairpin
loop are not affecting the structure of other regions of CA for example the
α4–α6 region. Taken together, these observations are consistent
with the involvement of much, if not all, of the surface of N-MLV CA in
recognition and binding by rhesus TRIM5α. Certainly the surfaces of the two
proteins appear to lie in sufficiently close proximity that the introduction of
charged residues in a variety of different positions prevents binding. An
extensive recognition surface fits well with distance measurements of the B30.2
domain from TRIMs closely related to TRIM5 [40], [41] that reveal that the
specificity determiningV1 and V3 regions are separated by about 30 Å, a
number very similar to that between the two sides of CA residues (see above).
However, attempts to identify regions of TRIM5α that bind specific CA
residues using TRIM5α chimeras such as those illustrated in Figure 5 have not met with
great success; rather it appears that well separated TRIM5α regions act in
combinatorial fashion to determine restriction specificity.

Comparison of the restriction factor binding sites on different
retroviruses

TRIM5α is capable of recognizing at least four genera of retroviruses despite
extensive differences in primary sequences of CA [23], [24], [25]. However CA's show
extensive structural conservation particularly in the core fold of helices
1–4 [42]. It thus appears likely that it is some shared
feature of overall structure that represents the target for TRIM5α rather
than direct sequence specific recognition alone. Loop exchange studies involving
different lentiviruses suggest that the primary target for the restriction
factor lies in the cyclophilin A binding loop, in a region analogous to the
originally defined Fv1 binding site of MLV [33], [34]. The importance of this
region is emphasized by the recent isolation of an HIV-1 escape mutant from
rhTRIM5α, V86M, by methods similar to those used here [43] and the roles of HIV-2
P120 and G116 in resistance to cynomolgus macaque TRIM5α [35], [44]. HIV-1 V86
and MLV N82 both lie just C-terminal to helix 4 while HIV-1 G116 and HIV-2 P120
lie close to position 110 of MLV. Further, domain swap experiments also suggest
a role for the β-hairpin region of SIV CA protein in the
evasion from rhTRIM5α-mediated restriction [36].

Based on these results and our success of producing and mapping
β-hairpin escape mutants of N-MLV that could evade
restriction by rhTRIM5α, it seemed logical to extend these studies and test
for commonality of binding by examining the restriction phenotypes of
β-hairpin mutants in HIV-1. Moreover, a comparison of the
CA-NtDs revealed that, although wider, the β1-hairpin-α6
cleft is also present in HIV-1 (Supplementary Figure S8).
Therefore, it might be expected that the introduction of bulky or charged side
chains into this channel would alter HIV susceptibility to TRIM5α in the
same way as in N-MLV. To this end, the studies presented in supplementary Figure S5
were undertaken. However they showed that viable single amino acid mutations in
the HIV-1 β-hairpin had no effect on restriction by
rhTRIM5α. These data suggest that the β-hairpin-α6
cleft is not a shared target for all viruses, implying that an as yet
unidentified binding region near the β-hairpin remains to be
discovered. In any event, it seems most likely that lentiviruses have an
extended TRIM5α binding site in analogous to that seen in MLV.

Human and rhesus TRIM5α restriction of MLV, the devil is in the
detail

The results of the initial passaging studies (Figure 1) together with phenotypic analysis
of our escape mutants (Figure
5) suggest that there are significant differences in the recognition
of N-MLV by rhesus and human TRIM5α. From the outset, restriction of N-MLV
by huTRIM5α was much more potent to the extent that we have been unable to
overcome N-MLV restriction by the hu-TRIM5α. Moreover, it is also refractory
to all but one of the changes isolated that allow escape from the rh-TRIM5α
(Table 1). The one
exception, E92K, was resistant to all restriction factors tested but showed a
significant reduction in titre. These observations suggested that the
interaction of huTRIM5α with N-MLV was either stronger and can elicit a more
powerful host cell response or that the CA-TRIM5α interface is different and
does not include the residues in the β-hairpin-α6 channel.
These observations are somewhat perplexing given the degree of similarity of the
TRIM5α B30.2 domains. However, it is notable that L10 and V116D mutations
have differential effects on restriction by TRIM5αs derived from other
species. For example V116D shows no restriction by orang-utan TRIM5α but is
restricted by sooty mangeby whereas L10W shows the converse sensitivity (Table 1). Further, of the
fourteen TRIM5αs we have previously examined only two, pigtailed macaque and
sooty mangabey, show identical restriction profiles against a panel of ten
different viruses [23], [24], [25].

Taken together, these variable determinants of restriction imply that TRIM5α
recognizes its targets in a manner significantly different from many
protein-protein interactions that involve a small number of highly conserved
residues, exemplified by the interaction betweens HIV-1 CA and the cellular
protein cyclophilin A [45]. Rather the interactions are predominately weak and
spread over a large surface. Avidity could then be provided by multivalent
binding to the regularly spaced CA molecules assembled on the viral core [26],
[27],
[28]. This
notion is further supported by recent structural data suggesting that formation
of a complementary TRIM5α lattice is actually a requirement for a productive
interaction of the restriction factor with capsid αassemblies
(Ganser-Pornillos et al, 2011). With the idea of lattice complementarity in mind
it is also interesting to note that although differences in conformation around
the top of Helix-6 between wt and L10W are only very small (∼1.2Å)
this region is centrally located in the extended restriction factor
binding-site. It is plausible that such small changes whilst not affecting
hexamer or CA lattice formation when propagated throughout an entire capsid
lattice could profoundly affect any lattice complementarity, altering
restriction factor susceptibility and resulting in viral escape from some
TRIM5αs. Whatever the case, retroviral pathogens comprise a wide range of
sequence divergent but structurally related assembled repeated capsid moieties
[42].
Perhaps weak interactions spread over a large binding surface are implicit in
the design of a defence mechanism intended to recognize these diverse
targets.

DNA

The cloning of human TRIM5α from TE671 and a chimeric human/rhesus macaque
TRIM5α (human RBCC domain fused with rhesus macaque B30.2 domain) have been
described previously [8], [24]. This chimeric human/rhesus macaque TRIM5α was
used throughout this study to generate and select escape mutants of N-MLV,
thereby allowing a direct comparison of interactions with the human and rhesus
macaque B30.2 domains, avoiding confounding effects of the coiled coil region
[16], [46], [47], and will
be referred to as rhesus TRIM5α. Generation of transduction vectors
expressing TRIM5α and Fv1 using the Gateway system was also described
previously [24], [48]. Single nucleotide mutations were introduced into the
capsid genes of Gag-Pol vector plasmids (pCIG3N and pCIG3B) and N-MLV provirus
plasmid (pWN41 [17]) by the QuickChange mutagenesis PCR protocol. Random
mutations were introduced at position 10 on MLV capsid by the QuickChange
mutagenesis PCR using the following primer pair: forward 5′- ggggggtaatggtcagNN(G/T)cagtactggccgttttc
-3′ and reverse 5′- gaaaacggccagtactg(C/A)NNctgaccattacccccc
-3′, where N indicates any nucleotide and G/T and C/A
indicate G or T and C or A, respectively. The sequences of the other primers
used to prepare mutants are available upon request. All introduced mutations
were verified by DNA sequencing.

Viruses

Single-cycle replication viruses were produced by transfection of VSV-G envelope
(pcz VSV-G), retroviral gag-pol (pCIG3N and pCIG3B for N- and B-tropic viruses,
respectively) and viral genomic plasmids (pczCFG2fEGFPf) into 293T cells by a
conventional calcium phosphate method as described previously [48] or using
Turbofect (Fermentas). Sixteen hours after transfection, cells were treated with
10 mM sodium butyrate for 6 hours, followed by replacement of the culture
supernatant with fresh growth medium. Virus-containing supernatant was harvested
48 hours after transfection, filtered through a 0.45 µm pore filter disk
and stored at −80°C until usage. Three stocks of replication competent
N-MLV were used. One (Stock A) was prepared from the culture supernatant of MDTF
cells transfected with N-MLV provirus (pWN41) followed by multiple (>20x)
passage of the transfected cells. The second (Stock B) was generated by
transfection of 293T cells with pWN41, transfer of the filtered supernatant to
MDTF cells followed by three cell passages. The third (Stock C) was unpassaged
supernatant of 293T cells transfected with pWN41. For transfection, pWN41 DNA
was digested with Hind III to separate the permuted provirus from the vector,
and purified viral DNA fragments were self-ligated with T4 DNA ligase at
16°C overnight. After purification, the ligated DNA was introduced into
MDTF/293T cells using a commercial transfection reagent according to the
manufacturer's instructions (TurboFect, Fermentas). Transfected cells were
re-fed with fresh medium after 16 hours; culture supernatants were harvested
after 48 hours.

Virus production in newly transfected/infected cells was monitored by protein
blotting of pelleted culture supernatants with monoclonal antibodies specific to
p12 and CA viral antigens (prepared from the culture supernatant of hybridomas
548 and R187 [ATCC]) or with a RT assay kit used according to the
manufacturer's instructions (C-type RT assay kit, Cavidi Tech, Sweden).

Mutant virus selection.

Target cells (MDTFs or rhTRIM5α expressing MDTFs or specified mixtures of
the two cell types) were seeded on a 6-well plate at 2×105
cells per well one day before virus inoculation. Replication competent N-MLV
was added to target cells at a titer equivalent to 514 mU of RT activity,
followed by washing cells three times with the growth medium 16 hours after
infection. The input virus was allowed to grow in the culture by passing
infected cells and virus spread was assessed by fluorescent staining. Once
the majority of restriction factor positive cells were infected with N-MLV,
the culture supernatant was harvested, filtered, and then stored at
−80°C until usage. The filtered supernatant was diluted 1∶10
with growth medium and inoculated onto fresh, restriction factor-expressing
cells. This virus passage cycle was repeated several times until the virus
spread rapidly in restriction factor positive cells.

FACS

Virus spread in infected cells was monitored by detection of an intracellular
virus antigen, p12. Infected cells were harvested by trypsinization and washed
with PBS, followed by fixation with 1.25% ice-cold paraformaldehyde in
PBS. All of the following procedures were performed on ice. Fixed cells were
washed once with permeabilization buffer (0.5 mg/ml digitonin and 1%
bovine serum albumin [BSA] in PBS), followed by incubation with
anti-p12 monoclonal antibody diluted in permeabilisation buffer. After washing
three times with wash buffer (5% Tween-20 and 1% BSA in PBS),
cells were then stained with anti-mouse IgG conjugated with AlexaFluor 594
(Invitrogen) diluted in permeabilisation buffer. After washing three times,
cells were re-suspended in 1.25% paraformaldehyde in PBS and analysed by
FACSCalibur within four hours.

Single-cycle replication experiments were performed as previously described [24], [48] using a
delivery vector encoding restriction factor and eYFP and a tester virus encoding
eGFP. Two colour FACS was carried out with a BD LSR II machine.

Sequencing of the capsid gene

Virus containing culture supernatant was harvested and filtered through a 0.45
µm-pore filter disk. The filtered supernatant was stored at
−80°C until usage. The supernatant was treated with MgCl2
and RNase-free DNase I at 37°C for 2 hours to degrade contaminating DNA.
DNase I-treated supernatant was subjected to viral RNA extraction using a
commercial kit (Viral RNA mini kit, QIAGEN). A single strand cDNA spanning the
capsid region was synthesized from viral RNA as a template with a primer
designed in the protease gene
(5′-gggttatcctgggttcaggggggggctcctgaccctg-3′) using a commercial kit
(Transcriptor High Fidelity cDNA synthesis kit, Roche). The cDNA synthesized was
used as a template to amplify viral capsid gene with a primer pair designed in
viral matrix (5′-gtctcgcctgcggggcaaaagag-3′) and protease (the same
as above) genes using PfuUltra high fidelity DNA polymerase (Stratagene). The
PCR product was gel purified, and nucleotide sequence was determined by direct
sequencing.

Fate-of-capsid assay

The fate-of-capsid assay was performed as previously described [38] with
minor modifications. MDTF cells expressing TRIM5α (5×106
cells in a 80 cm2 tissue culture flask) were pre-plated one day
before assay. Cells were inoculated with freshly prepared, chilled virus (14 ml
per flask with 5 ug/ml polybrene) from the filtered culture supernatant of
transiently transfected 293T cells. After incubation on ice for 30 minutes,
cells were incubated at 37°C for 4 hours. Cells were then washed three times
with ice-cold PBS and treated with 1 ml of 7 mg/ml pronase at room temperature
for 5 minutes. Cells were re-suspended in ice-cold growth medium and washed
three times with ice-cold PBS. Cells were re-suspended in 2.5 ml of hypotonic
lysis buffer [10 mM Tris-HCl (pH 8.0), 10 mM KCl, 1 mM EDTA and protease
inhibitor cocktail] and incubated on ice for 15 minutes. Swollen cells were
lysed in a 7 ml-Dounce homogenizer with a ‘tight’ pestle (15 gentle
strokes) and cell lysates cleared by centrifugation at 4°C and 1,850 xg for
3 minutes. Cleared cell extracts (2 ml) were layered over 50% sucrose
cushions prepared in PBS and centrifuged at 4°C and 125,000 xg for 2 hours
in a Beckman SW41 rotor and 100 µl of the cell extract was collected as a
‘input’ fraction. After the centrifugation, 100 µl of the
topmost portion of the supernatant was collected as a ‘soluble’
fraction. The pellet was re-suspended in 100 µl 1×SDS sample loading
buffer (‘pellet’ fraction). Samples were separated on acrylamide
gels and blotted for CA as described above.

Protein crystallisation and structure determination

The CA N-terminal domain of N-MLV(L10W) was crystallised by sitting-drop vapour
diffusion using a reservoir solution containing 24% PEG-8000, 0.1M
phosphate citrate, pH 5.0 mixed with an equal volume of protein solution (14
mg/ml). Prior to data collection, crystals were transferred into a
cryoprotectant of reservoir solution containing 20% glycerol and flash
frozen in liquid nitrogen. X-ray diffraction data were collected at 100° K
on a Rigaku Micromax-007HF with a Raxis-IV detector to a resolution of
2.0Å and processed using the HKL program package [49]. Crystals belonged to
the space group C2 with the cell dimensions
a = 71.9Å, b = 33.6Å,
c = 59.3Å,
β = 111.9°.

The structure of N-MLV(L10W) was determined by molecular replacement using a
monomer of N-MLV CA-NtD (1U7K) as a search model in PHASER [50]. A single molecule was
present in the asymmetric unit (Z-score 12.3, LLG 590). The model was rebuilt
using Arp/Warp (Morris et al., 2003) followed by iterative rounds of refinement
and model building in PHENIX [51] and COOT [52]. Nine TLS groups were
included in final round of refinement as determined by TLSMD [53]. The
structure was refined to a final Rwork/Rfree of
17.4%/24.4% respectively.

Coordinates

The coordinates and structure factors of N-MLV(L10W) have been deposited in the
Protein Data Bank under accession number 2y4z.

Restriction by rhesus macaque TRIM5α of N-MLV carrying different amino
acids at CA position 10. A variety of amino acids were introduced at
position 10 of CA by random mutagenesis. Unique isolates were tested for
growth in the presence (solid line and filled squares) or absence (dotted
line and open diamonds) of rhesus macaque TRIM5α. One representative
result from three independent experiments is shown. These data are
summarized in Table
2.

Titration curves of selected N-MLV escape mutants in the presence of various
primate TRIM5α. Chimeric TRIM5α constructs expressing the human RBCC
domain fused with different primate B30.2 domains [24] were used to test
restriction of wild type and L10W, L10H, L10K and V116D escape mutant virus.
Titration curves in the presence of ape, old world monkey and new world
money TRIM5α are shown in green (left column), red (middle column) and
blue (right column), respectively. One representative result is shown from
three independent experiments. These data are summarized in Table 3.

Titration curves of N-MLV escape mutants in the presence of chimeric
TRIM5α. Titration curves in the presence of human-rhesus TRIM5α
chimeras are shown in the left column and those in the presence of
rhesus-human TRIM5α chimera are shown in the right column. One
representative result from three independent experiments is shown. These
data are summarized in Figure
5.

Titration curves of HIV-1 with lysine-substitutions within the CA
β-hairpin loop. Lysine substitutions were introduced at amino acid
positions 5 through 12 of HIV-1 CA as well as random amino acid mutations at
position 10. These were tested for growth in the presence (solid line and
filled squares) or absence (dotted line and open diamonds) of rhTRIM5α.
Data for the I6K, G8K and H12K mutants showed much reduced infectivity and
are not shown. One representative result from two independent experiments is
shown.

Stereo 2Fo-Fc electron density. Maps contoured at 1.0σ around the region
of N-MLV containing either L10 (1U7K) in panel A or W10 (2Y4Z) in panel B
are shown. The protein main-chain is shown in cartoon representation,
residue side chains are shown in stick representation, L10 and W10 are
highlighted in red.

Mutation R112A abolishes relief of TRIM 5α restriction by L10W. Single
cycle experiments to examine the effect of the R112A mutation on restriction
of wt and L10W N-MLV by TRIM5α. Infection was tested in the presence
(filled squares) or absence (open diamonds) of rhTRIM5α. Panel A, N-MLV
carrying the L10W mutation; Panel B. N-MLV with R112A; Panel C, N-MLV with
L10W and R112A. One representative result from three independent experiments
is shown.

The β1-β2-α6 cleft in HIV-1 CA-NtD. Side (lower) and top (upper)
views of the HIV-1 CA-NtD structure are displayed (PDB ID 1M9C). The protein
backbone is shown in cartoon representation together with the
semi-transparent molecular surface. Residues in the amino-terminal
β-hairpin that were mutated in order to look for escape
from rhesus TRIM5α restriction are shown in stick representation. The
cleft between β1-β2 and helix 5′ at
the top of a6 is much wider in HIV than in MLV; cf. Figure 6.

doi:10.1371/journal.ppat.1002011.s008

(TIF)

Author Contributions

Conceived and designed the experiments: SO IAT JPS. Performed the
experiments: SO DCG MWY KHD. Analyzed the data: SO DCG MWY KHD IAT JPS.
Wrote the paper: SO IAT JPS.